The present study aimed to investigate which of two commonly performed running interval sessions elicited the greatest magnitude of and time spent with elevated muscle deoxygenation in trained middle-distance runners. Thirteen trained middle-distance runners (22.4 ± 3.2 y; 63.1 ± 10.9 kg; n = 9 males) participated in the study. Subjects completed a field-based incremental running test and two interval sessions. The interval sessions comprised a 6 × 1 km and a 15 × 400 m interval session, both with 1 min passive recovery periods. Both sessions were implemented with the aim of achieving the maximal sustainable pace for each repetition, while mean speed, heart rate, RPE, blood lactate concentration and muscle deoxygenation responses were monitored. Mean speed during the interval repetitions was significantly higher during the 400 m intervals (~ 5.63 ± 0.35 m·s−1 vs ~ 5.30 ± 0.28 m·s−1; p < 0.001). Both the peak magnitude of muscle deoxygenation (absolute difference ± CI 3.42 ± 2.23%; p = 0.006) and the time spent with values > 60% peak muscle deoxygenation (83.5 ± 66.4 s; p = 0.02) were significantly greater during the 400 m intervals, while the time spent with a heart rate > 90% peak heart rate was significantly longer during the 1 km interval session (570 ± 143, p < 0.001). Despite this, there was no difference in RPE, blood lactate concentration or peak heart rate between sessions. These findings suggest that 1 km intervals may preferentially target central physiologic responses while 400 m intervals may elicit greater peripheral physiological responses in trained middle-distance runners.